Super-resolved analysis of colocalization between replication and transcription along the cell cycle in a model of oncogene activation.

To understand how oncogenes affect genome organization, it is essential to visualize fundamental processes such as DNA replication and transcription at high resolution in intact cells. At the same time, it is important to determine the progression of the cell along the cell cycle, as cell cycle regulation is crucial for the control of cell proliferation and oncogenesis. Here, we present a super-resolution imaging-based method to analyze single cell nuclei sorted according to specific phases of the cell cycle.

From Cell Populations to Molecular Complexes: Multiplexed Multimodal Microscopy to Explore p53-53BP1 Molecular Interaction.

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory.

A DNA damage response-like phenotype defines a third of colon cancers at onset.

Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportunities, besides DNA hypermutating cases; thus, both new targets or broadening existing strategies for personalized intervention are of interest. Routinely processed material from 246 untreated COADs with clinical follow-up was probed for evidence of DNA damage response (DDR), that is, the gathering of DDR-associated molecules at discrete nuclear spots, by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (γH2AX, pCHK2, and pNBS1). We also tested the cases for type I interferon response, T-lymphocyte infiltration (TILs), and mutation mismatch repair defects (MMRd), known to be associated with defects of DNA repair.

Correlative Multi-Modal Microscopy: A Novel Pipeline for Optimizing Fluorescence Microscopy Resolutions in Biological Applications.

The modern fluorescence microscope is the convergence point of technologies with different performances in terms of statistical sampling, number of simultaneously analyzed signals, and spatial resolution. However, the best results are usually obtained by maximizing only one of these parameters and finding a compromise for the others, a limitation that can become particularly significant when applied to cell biology and that can reduce the spreading of novel optical microscopy tools among research laboratories. Super resolution microscopy and, in particular, molecular localization-based approaches provide a spatial resolution and a molecular localization precision able to explore the scale of macromolecular complexes in situ.

Automated multimodal fluorescence microscopy for hyperplex spatial-proteomics: Coupling microfluidic-based immunofluorescence to high resolution, high sensitivity, three-dimensional analysis of histological slides.

multiplexing analysis and transcriptomics are now providing revolutionary tools to achieve the comprehension of the molecular basis of cancer and to progress towards personalized medicine to fight the disease. The complexity of these tasks requires a continuous interplay among different technologies during all the phases of the experimental procedures. New tools are thus needed and their characterization in terms of performances and limits is mandatory to reach the best resolution and sensitivity.

Alterations induced by the PML-RARα oncogene revealed by image cross correlation spectroscopy.

The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. To understand how oncogenes affect chromatin architecture, it is important to visualize fundamental processes such as DNA replication and transcription in intact nuclei and quantify the alterations of their spatiotemporal organization induced by oncogenes. Here, we apply superresolution microscopy in combination with image cross correlation spectroscopy to the U937-PR9 cell line, an in vitro model of acute promyelocytic leukemia that allows us to activate the expression of the PML-RARα oncogene and analyze its effects on the spatiotemporal organization of functional nuclear processes.

From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action.

53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e.

Novel Tools to Measure Single Molecules Colocalization in Fluorescence Nanoscopy by Image Cross Correlation Spectroscopy.

Super Resolution Microscopy revolutionized the approach to the study of molecular interactions by providing new quantitative tools to describe the scale below 100 nanometers. Single Molecule Localization Microscopy (SMLM) reaches a spatial resolution less than 50 nm with a precision in calculating molecule coordinates between 10 and 20 nanometers. However new procedures are required to analyze data from the list of molecular coordinates created by SMLM.

LSD1-directed therapy affects glioblastoma tumorigenicity by deregulating the protective ATF4-dependent integrated stress response.

Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, poor blood-brain barrier penetration, and resistance to therapy highlight the need for new targets and clinical treatments. A step toward clinical translation includes the eradication of GBM tumor-initiating cells (TICs), responsible for GBM heterogeneity and relapse. By using patient-derived TICs and xenograft orthotopic models, we demonstrated that the selective lysine-specific histone demethylase 1 inhibitor DDP_38003 (LSD1i) is able to penetrate the brain parenchyma in vivo in preclinical models, is well tolerated, and exerts antitumor activity in molecularly different GBMs.

Evaluation of sted super-resolution image quality by image correlation spectroscopy (QuICS).

Quantifying the imaging performances in an unbiased way is of outmost importance in super-resolution microscopy. Here, we describe an algorithm based on image correlation spectroscopy (ICS) that can be used to assess the quality of super-resolution images. The algorithm is based on the calculation of an autocorrelation function and provides three different parameters: the width of the autocorrelation function, related to the spatial resolution; the brightness, related to the image contrast; the relative noise variance, related to the signal-to-noise ratio of the image.

Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy.

Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured illumination microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct an SR image.

Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS).

Since the introduction of super-resolution microscopy, there has been growing interest in quantifying the nanoscale spatial distributions of fluorescent probes to better understand cellular processes and their interactions. One way to check if distributions are correlated or not is to perform colocalization analysis of multi-color acquisitions. Among all the possible methods available to study and quantify the colocalization between multicolor images, there is image cross-correlation spectroscopy (ICCS).

Antibodies validated for routinely processed tissues stain frozen sections unpredictably.

Antibody validation for tissue staining is required for reproducibility; criteria to ensure validity have been published recently. The majority of these recommendations imply the use of routinely processed (formalin-fixed, paraffin-embedded) tissue. We applied to lightly fixed frozen sections a panel of 126 antibodies validated for formalin-fixed, paraffin-embedded tissue with extended criteria.

The Adaptive and Innate Immune Cell Landscape of Uterine Leiomyosarcomas.

Reactivation of the anti-tumor response has shown substantial progress in aggressive tumors such as melanoma and lung cancer. Data on less common histotypes are scanty. Immune checkpoint inhibitor therapy has been applied to few cases of uterine leiomyosarcomas, of which the immune cell composition was not examined in detail.

Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy.

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions.

Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations.

It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5' splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs.

Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures.

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image.

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections.

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities.

RNAi screens identify CHD4 as an essential gene in breast cancer growth.

Epigenetic regulation plays an essential role in tumor development and epigenetic modifiers are considered optimal potential druggable candidates. In order to identify new breast cancer vulnerabilities and improve therapeutic chances for patients, we performed in vivo and in vitro shRNA screens in a human breast cancer cell model (MCF10DCIS.com cell line) using epigenetic libraries.

Cell cycle-dependent resolution of DNA double-strand breaks.

DNA double strand breaks (DSBs) elicit prompt activation of DNA damage response (DDR), which arrests cell-cycle either in G1/S or G2/M in order to avoid entering S and M phase with damaged DNAs. Since mammalian tissues contain both proliferating and quiescent cells, there might be fundamental difference in DDR between proliferating and quiescent cells (or G0-arrested). To investigate these differences, we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs in asynchronously proliferating, G0-, or G1-arrested cells.

Molecular investigation of coexistent chronic myeloid leukaemia and peripheral T-cell lymphoma - a case report.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm underlain by the formation of BCR-ABL1 - an aberrant tyrosine kinase - in the leukaemic blasts. Long-term survival rates in CML prior to the advent of tyrosine kinase inhibitors (TKIs) were dismal, albeit the incidence of secondary malignancies was higher than that of age-matched population. Current figures confirm the safety of TKIs with conflicting data concerning the increased risk of secondary tumours.

Extracellular vesicle-mediated transfer of CLIC1 protein is a novel mechanism for the regulation of glioblastoma growth.

Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential.

The Combination of the PARP Inhibitor Rucaparib and 5FU Is an Effective Strategy for Treating Acute Leukemias.

The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most patients, drawing attention to the need of new therapeutic strategies. In the last decade the anticancer potential of poly ADP-ribose polymerase (PARP) inhibitors became apparent and now several PARP inhibitors are being developed to treat various malignancies. So far, the usage of PARP inhibitors has been mainly focused on the treatment of solid tumors and not too much about their efficacy on leukemias is known.

Confocal microscopy for high-resolution and high-content analysis of the cell cycle.

Optical fluorescence microscopy offers a wide range of technological solutions to address many questions in biomedical research. Spatial resolution has been greatly improved by the use of confocal microscopes, providing a 3-D analysis of the intracellular space. Automation has contributed to make confocal analysis available for high-content image cytometry studies.